Disruption of Oligomerization and Dehydroalanine Formation as Mechanisms for ClpP Protease Inhibition
Disruption of Oligomerization and Dehydroalanine Formation as Mechanisms for ClpP Protease Inhibition
复制标题
DOI：
10.1021/ja4082793
复制
发表时间：
2014-01-29
影响因子：
15
通讯作者：
Sieber, Stephan A.
中科院分区：
文献类型：
--
作者：
Gersch, Malte;Kolb, Roman;Sieber, Stephan A.
目前临床上使用的蛋白酶抑制剂超过100种，其中大多数采用活性位点的封闭作为其抑制模式。在蛋白酶药物靶点中，有几种酶的正确多聚体组装对其活性至关重要，如蛋白酶体和HIV蛋白酶。在这里，我们提出了一种新的蛋白酶抑制机制，依赖于活性位点定向的小分子，分解蛋白酶复合物。我们显示了这种机制在ClpP蛋白酶家族中的适用性，ClpP蛋白酶家族的成员是十四聚体丝氨酸蛋白酶，并作为几种细胞过程的调节剂，包括稳态和毒力。化合物以亚化学计量方式与ClpP结合触发完全无活性的七聚体的形成。此外，我们报告的选择性β-磺内酰胺诱导脱氢丙氨酸形成的活性位点丝氨酸。该反应通过磺酰化和随后的消除进行，从而消除催化电荷中继系统。通过质谱和晶体学确认脱氢丙氨酸的身份。基于活性的蛋白质分析实验表明，在活的S.金黄色葡萄球菌细胞。总的来说，这些发现扩展了我们对多组分蛋白酶抑制的看法，到目前为止，这主要依赖于活性位点的阻断或调节变构位点的占据。
Over 100 protease inhibitors are currently used in the clinics, and most of them use blockage of the active site for their mode of inhibition. Among the protease drug targets are several enzymes for which the correct multimeric assembly is crucial to their activity, such as the proteasome and the HIV protease. Here, we present a novel mechanism of protease inhibition that relies on active-site-directed small molecules that disassemble the protease complex. We show the applicability of this mechanism within the ClpP protease family, whose members are tetradecameric serine proteases and serve as regulators of several cellular processes, including homeostasis and virulence. Compound binding to ClpP in a substoichiometric fashion triggers the formation of completely inactive heptamers. Moreover, we report the selective beta-sultam-induced dehydroalanine formation of the active site serine. This reaction proceeds through sulfonylation and subsequent elimination, thereby obliterating the catalytic charge relay system. The identity of the dehydroalanine was confirmed by mass spectrometry and crystallography. Activity-based protein profiling experiments suggest the formation of a dehydroalanine moiety in living S. aureus cells upon beta-sultam treatment. Collectively, these findings extend our view on multicomponent protease inhibition that until now has mainly relied on blockage of the active site or occupation of a regulatory allosteric site.